1 Project Summary 2 This project focuses on the chloroplast as central player in the generation of immune signals and the 3 regulation of programmed cell death (PCD) required for innate immune responses against pathogen infection. 4 Although mitochondria play a central role during mammalian PCD, emerging evidence suggests that in plants 5 chloroplasts have a critical function in executing localized PCD that limits pathogen spread. Chloroplasts in 6 addition to being involved in the generation of immune signals, such as reactive oxygen species (ROS) and the 7 defense hormone salicylic acid (SA), also participate directly in the recognition of pathogens. Interestingly, 8 chloroplasts dynamically change their morphology during immune responses and send out stroma-filled tubular 9 projections called stromules. These induced stromules use the cytoskeleton to extend and then anchor to the 10 nucleus, which facilitate perinuclear clustering of chloroplasts and transport of chloroplast-generated hydrogen 11 peroxide (H2O2) ROS and defense proteins to the nucleus. The overall goal of this application is to use a 12 combination of novel cell biology, genetics, proteomics and computational approaches to unravel the 13 mechanistic basis of stromule formation and their role in driving perinuclear chloroplast clustering and 14 subsequent release of retrograde immune signals from chloroplasts to nuclei. Specifically, Aim 1 will identify 15 and characterize proteins required for stromule formation and stromule-directed chloroplast movement. This 16 includes the kinesin motor required for extension and proteins associated with the outer envelope of stromules 17 that may drive stromule initiation or regulation. An unbiased forward genetic screen will be conducted to 18 identify other stromule specific components. Aim 2 will investigate immune signals required for stromule 19 induction and the release of H2O2 as a specific retrograded chloroplast signal. The role of stromules in 20 amplification of immune signaling consisting of SA and H2O2 signaling will be examined. The relationship of 21 different H2O2 sources, organelle movement and PCD during immune responses will be studied to determine 22 how H2O2 propagates extracellularly to intracellular sources to regulate innate immune responses. The function 23 of stromules and chloroplast positioning during H2O2 signal propagation will be examined using the stromule- 24 specific mutants characterized in Aim 1. Aim 3 will focus on how pathogen effectors affect stromules, 25 organelle, and cytoskeleton dynamics as a virulence strategy. In addition, mechanistic basis of how three 26 effectors disrupt stromules and organelle dynamics will be determined. Understanding the role of different 27 organelles during PCD and innate immunity will provide a unified mechanistic basis of cell death and cell 28 survival process that occur in response to infectious pathogens. The results from our model systems will 29 impact broadly on understanding of organelle-to-nuclear communication that influence innate immunity against 30 infectious diseases.